Pulmonary epithelium is devided into three histopathologically distinct compartments: bronchi, bronchioli, and alveoli. Our long term goal is to characterize the morphological changes, aberrant cellular differentiation and genetic damage associated with premalignant changes and carcinogenesis in each compartment. A. Peripheral airway cell (PAC) and neuroendocrine (NE) differentiation We have shown that field cancerization in human lung is associated with alterations in the expression patterns of PAC and NE markers. We are now developing experimental models in which the pulmonary changes parallel those seen in man. In response to carcinogen, there is a marked decrease in the expression of Clara cell specific protein (CC10), a PAC marker of progenitor cells in non-neoplastic and neoplastic airway epithelium in hamsters and mice. This is followed by NE cell hyperplasia and tumor formation later on. In order to see if reduced CC10 expression actually contributes to carcinogenesis in vivo, we exposed CC10 knockout mice to the tobacco-specific nitrosamine NNK. We observed more lung tumors in CC10 deficient animals than their wild type counterparts. This was accompanied by hyperproliferation in airway epithelium and activation of ras/MAP kinase pathway. The outcome is in concert with our in vitro experiments, in which overexpression of CC10 in lung cancer cells and immortalized bronchial epithelial cells antagonized neoplastic phenotype and affected tumor progression. Approximately one third of all human lung cancers are characterized by NE differentiation. We have shown that a neural transcription factor from Drosophila, achaete-schute homolog-1 is expressed at high levels in NE lung cancers and is essential for NE differentiation in neoplastic and non-neoplastic lung. Constitutive expression of this transcription factor under CC10 promoter caused marked bronchialization of alveoli, which provides a model for a potential premalignant lesion for human lung adenocarcinomas. We have demonstrated that this lesion progresses through proliferation and resistance to apoptosis. We are currently assessing the molecular mechanisms in vitro using cell lines and siRNAs of achaete-schute homolog-1. We have also shown that constitutive expression of achaete-schute homolog-1under CC10 promoter together with SV40 caused massive tumors with NE differentiation, which provides a model for a clinically important subset of human non-small cell lung cancers with NE features. Using cDNA and oligo microarray technology we have identified differentially expressed genes in this tumor model, and are currently characterizing their potential functional role in NE carcinognesis. B. Mouse model for human small cell lung cancer (SCLC) The prototype of human NE lung cancers is small cell lung cancer (SCLC), the most common and virulent of all NE cancers in man. One hundred percent human SCLCs contain alterations of RB1 and/or P53 tumor suppressor genes. We established a mouse model by conditional inactivation of these genes in mouse lung epithelial cells. Mice carrying conditional alleles for both RB1 and P53 developed highly aggressive lung tumors with striking similarities with human SCLC in their morphology, immunophenotype and extrapulmonary metastatic spread. In our model, inactivation of both RB1 and p53 was a prerequisite for the pathogenesis of SCLC. We are now studying the role of lung specific transcription factors and cell cycle abnormalities in the mouse SCLC model and precursor lesions. C. Generating a mouse model for squamous cell carcinogenesis in the lung. Squamous cell carcinoma composes approximately 25% of all lung cancers in the United States. It is a central tumor arising from bronchi, and the only human lung carcinoma for which the precursor lesions are well established. However, squamous cell carcinoma in mouse is rare, and there is a critical need to have a good pre-clinical model for chemoprevention studies. We will establish a mouse model for lung squamous cell carcinoma. We postulate that keratin 5/14 (K5/K14) containing basal cells that are needed for the formation of squamous cell carcinomas. We have shown that they are scanty or non-existent in mouse intrapulmonary airways. We propose that constitutive expression of K5 or K14 under the CC10 promoter will allow squamous differentiation in the airway epithelium, and can be used as the basis for generation of squamous cell carcinomas. So far we have prepared the construct, and initiated the production of transgenic mice. We are also generating in vitro cell lines that express K14 under CC10 promoter for functional studies. The significance of this project is that it will generate a preclinical model for testing novel chemotherapeutic agents. The significance of this research is that the results will provide a rational basis for early detection and molecular targets for therapeutic interventions for human lung carcinogenesis by identifying specific markers and pathways as well as distinct models of multistep epithelial carcinogenesis.